Tick-borne encephalitis virus capsid protein induces translational shutoff as revealed by its structural-biological analysis.

Tick-borne encephalitis virus (TBEV) is the most medically relevant tick-transmitted Flavivirus in Eurasia, targeting the host central nervous system and frequently causing severe encephalitis. The primary function of its capsid protein (TBEVC) is to recruit the viral RNA and form a nucleocapsid. Additional functionality of Flavivirus capsid proteins has been documented, but further investigation is needed for TBEVC. Here, we show the first capsid protein 3D structure of a member of the tick-borne flaviviruses group. The structure of monomeric Δ16-TBEVC was determined using high-resolution multidimensional NMR spectroscopy. Based on natural in vitro TBEVC homodimerization, the dimeric interfaces were identified by hydrogen deuterium exchange mass spectrometry (MS). Although the assembly of flaviviruses occurs in endoplasmic reticulum-derived vesicles, we observed that TBEVC protein also accumulated in the nuclei and nucleoli of infected cells. In addition, the predicted bipartite nuclear localization sequence in the TBEVC C-terminal part was confirmed experimentally, and we described the interface between TBEVC bipartite nuclear localization sequence and import adapter protein importin-alpha using X-ray crystallography. Furthermore, our coimmunoprecipitation coupled with MS identification revealed 214 interaction partners of TBEVC, including viral envelope and nonstructural NS5 proteins and a wide variety of host proteins involved mainly in rRNA processing and translation initiation. Metabolic labeling experiments further confirmed that TBEVC and other flaviviral capsid proteins are able to induce translational shutoff and decrease of 18S rRNA. These findings may substantially help to design a targeted therapy against TBEV.

Self-Patterning Polyelectrolyte Multilayer Films: Influence of Deposition Steps and Drying in a Vacuum.

Typically, laterally patterned films are fabricated by lithographic techniques, external fields, or di-block copolymer self-assembly. We investigate the self-patterning of polyelectrolyte multilayers, poly(diallyldimethylammonium) (PDADMA)/poly(styrenesulfonate) (PSS)short. The low PSS molecular weight (Mw(PSSshort) = 10.7 kDa) is necessary because PSSshort is somewhat mobile within a PDADMA/PSSshort film, as demonstrated by the exponential growth regime at the beginning of the PDADMA/PSSshort multilayer build-up. No self-patterning was observed when the PDADMA/PSS film consisted of only immobile polyelectrolytes. Atomic force microscopy images show that self-patterning begins when the film consists of seven deposited PDADMA/PSSshort bilayers. When more bilayers are added, the surface ribbing evolved into bands, and circular domains were finally observed. The mean distance between the surface structures increased monotonously with the film thickness, from 70 to 250 nm. Scanning electron microscopy images showed that exposure to vacuum resulted in thinning of the film and an increase in the mean distance between domains. The effect is weaker for PSSshort-terminated films than for PDADMA-terminated films. The mechanism leading to domain formation during film build-up and the effect of post-preparation treatment are discussed.

Stopping the Mobile Robotic Vehicle at a Defined Distance from the Obstacle by Means of an Infrared Distance Sensor.

The article deals with the creation of a program for stopping an autonomous robotic vehicle Robotino® 4. generation at a defined distance from an obstacle. One of the nine infrared distance sensors located on the frame of the robotic vehicle in the front part of the frame is used for this application task. The infrared distance sensor characteristic is created from the measured experimental data, which is then linearized in the given section. The main aim of the experiment is to find such an equation of a line that corresponds to the stopping of a robotic vehicle with a given accuracy from an obstacle. The determined equation of the line is applied to the resulting program for autonomous control of the robotic vehicle. This issue is one of the many tasks performed by AGV in the industry. The introduction of AGVs into the industry is one of the many possibilities within Industry 4.0.

Proteome Profiling of PMJ2-R and Primary Peritoneal Macrophages.

In vitro models are often used for studying macrophage functions, including the process of phagocytosis. The application of primary macrophages has limitations associated with the individual characteristics of animals, which can lead to insufficient standardization and higher variability of the obtained results. Immortalized cell lines do not have these disadvantages, but their responses to various signals can differ from those of the living organism. In the present study, a comparative proteomic analysis of immortalized PMJ2-R cell line and primary peritoneal macrophages isolated from C57BL/6 mice was performed. A total of 4005 proteins were identified, of which 797 were quantified. Obtained results indicate significant differences in the abundances of many proteins, including essential proteins associated with the process of phagocytosis, such as Elmo1, Gsn, Hspa8, Itgb1, Ncf2, Rac2, Rack1, Sirpa, Sod1, C3, and Msr1. These findings indicate that outcomes of studies utilizing PMJ2-R cells as a model of peritoneal macrophages should be carefully validated. All MS data are deposited in ProteomeXchange with the identifier PXD022133.

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio.

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

Gas-aggregated Ag nanoparticles for detection of small molecules using LDI MS.

Several reports demonstrate that silver nanomaterials can serve as surface-assisted laser desorption ionization mass spectrometry (SALDI MS) substrates for low molecular weight analytes. Substrate with tailored silver nanostructures, primarily representing the upmost layer of the bulk, i.e., occurring beneath the analyzed medium, limits the use of silver only for desorption enhancement; the charge transfer progresses through atoms from the absorbing analyte or an additional matrix (resulting in the formation of analyte/hydrogen, sodium, or potassium adducts in the most cases). In the presented approach, we utilize a homogeneous layer of silver nanoparticles, prepared under low-pressure conditions, deposited onto a dried analyte. We demonstrate that the nanoparticle layer can fully replace a matrix typically used for the detection of small molecules by laser desorption/ionization mass spectrometry-based technique (LDI MS) and can be applied to the already prepared samples. Various chemical species were detected as [M + Ag]+ adduct ions employing the proposed technique. The normalized signal of the analyte/silver adduct can be utilized to characterize a quantitative presence of analytes on the surface similar to signal-to-noise value, here demonstrated by the detection of trimethoprim molecule. This study also includes a detailed description of additional features one needs to take into account, such as a formation of [Mx + Agy]+ adducts, presence of silver ions (can be used for m/z calibration), analyte fragmentation, and influence of deposited nanoparticles quantity on the signal intensity. Graphical abstract.

Protective role of antifreeze proteins on sterlet (Acipenser ruthenus) sperm during cryopreservation.

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 µg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85 ± 4% motility and 160 ± 2 µm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44 ± 9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 µg/mL of AFPIII (58 ± 14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5 ± 6% live cells, while the cryopreserved sperm only contained 26.6 ± 14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 µg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 µg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.

Protein profile of seminal plasma and functionality of spermatozoa during the reproductive season in the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

The purpose of the study was to analyze seminal plasma composition, sperm production, and sperm motility over the course of the spawning season in the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). The highest mean percent of motile sperm (carp, 98.3 ± 1.6%; rainbow trout, 92.8 ± 5.6%) and highest spermatozoon velocity (carp, 286.3 ± 7.8 µm sec-1 ; rainbow trout, 245.3 ± 8.3 µm sec-1 ) were observed in the middle phase of the spawning period, at 5 sec post-activation. Sperm volume and concentration increased in the early spawning period, then decreased significantly toward the end of the season in both species. Seminal plasma osmolality was 262-270 mOsmol kg-1 from carp and 232-248 mOsmol kg-1 from rainbow trout, with little variation over the spawning period. Protein concentration in carp seminal plasma was stable throughout the reproductive season, whereas the highest protein level (2.1 ± 0.3 mg mL-1 ) in rainbow trout was observed in the middle phase of the spawning period. Seminal plasma composition in both species was analyzed using two-dimensional polyacrylamide gel electrophoresis combined with mass spectrometry. Thirteen differentially expressed protein spots in carp seminal plasma and sixteen spots in rainbow trout seminal plasma varied over the course of the reproductive season. The unique proteins identified are involved in the regulation of spermatogenesis, sperm motility, maintenance of sperm membrane lipid stability, and antioxidant protection. These results provide a deeper understanding of the role of fish seminal plasma proteins as well as a list of novel markers of sperm quality. Mol. Reprod. Dev. 83: 968-982, 2016 © 2016 Wiley Periodicals, Inc.

Expression of a second open reading frame present in the genome of tick-borne encephalitis virus strain Neudoerfl is not detectable in infected cells.

A short upstream open reading frame (uORF) was recently identified in the 5' untranslated region of some tick-borne encephalitis virus (TBEV) strains. However, it is not known if the peptide encoded by TBEV uORF (TuORF) is expressed in infected cells. Here we show that TuORF forms three phylogenetically separated clades which are typical of European, Siberian, and Far-Eastern TBEV subtypes. Analysis of selection pressure acting on the TuORF area showed that it is under positive selection pressure. Theoretically, TuORF may code for a short hydrophobic peptide embedded in a biological membrane. However, expression of TuORF was detectable neither by immunoblotting in tick and mammalian cell lines infected with TBEV nor by immunofluorescence in TBEV-infected mammalian cell lines. These results support the idea that TuORF is not expressed in TBEV-infected cell or expressed in undetectably low concentrations. Therefore we can assume that TuORF has either minor or no biological role in the TBEV life cycle.

Characterization of proteolytic and anti-proteolytic activity involvement in sterlet spermatozoon maturation.

In sturgeon, the acquisition of the potential for motility activation called spermatozoon maturation takes place outside testes. This process can be accomplished in vitro by pre-incubation of immature testicular spermatozoa in seminal fluid collected from fully mature Wolffian duct sperm. Addition of trypsin inhibitor to the pre-incubation medium disrupts spermatozoon maturation. There are no available data for the role of proteolysis regulators in fish spermatozoon maturation, while their role is recognized in mammalian sperm maturation. The present study evaluated the involvement of seminal fluid proteases and anti-proteolytic activity in the sterlet spermatozoon maturation process. Casein and gelatin zymography and quantification of amidase and anti-proteolytic activity were conducted in sturgeon seminal fluid from Wolffian duct sperm and seminal fluid from testicular sperm, along with spermatozoon extracts from Wolffian duct spermatozoa, testicular spermatozoa, and testicular spermatozoa after in vitro maturation. We did not find significant differences in proteolytic profiles of seminal fluids from Wolffian duct sperm and ones from testicular sperm. Zymography revealed differences in spermatozoon extracts: Wolffian duct spermatozoon extracts were characterized by the presence of a broad proteolytic band ranging from 48 to 41 kDa, while testicular spermatozoon extracts did not show such activity until after in vitro maturation. The differences in amidase activity coincided with these results. It may not be the levels of proteolytic and anti-proteolytic activity per se, but the alterations in their interactions triggering a cascade of signaling events, that is crucial to the maturation process.

Aortic valve performance after remodelling versus reimplantation in a propensity-matched comparison.

OBJECTIVES: Both aortic root remodelling and aortic valve (AV) reimplantation have been used for valve-sparing root replacement in patients with aortic root aneurysm with or without aortic regurgitation. There is no clear evidence to support one technique over the another. This study aimed to compare remodelling with basal ring annuloplasty versus reimplantation on a multicentre level with the use of propensity-score matching. METHODS: This was a retrospective international multicentre study of patients undergoing remodelling or reimplantation between 2010 and 2021. Twenty-three preoperative covariates (including root dimensions and valve characteristics) were used for propensity-score matching. Perioperative outcomes were analysed along with longer-term freedom from AV reoperation/reintervention and other major valve-related events. RESULTS: Throughout the study period, 297 patients underwent remodelling and 281 had reimplantation. Using propensity-score matching, 112 pairs were selected and further compared. We did not find a statistically significant difference in perioperative outcomes between the matched groups. Patients after remodelling had significantly higher reintervention risk than after reimplantation over the median follow-up of 6 years (P = 0.016). The remodelling technique (P = 0.02), need for decalcification (P = 0.03) and degree of immediate postoperative AV regurgitation (P < 0.001) were defined as independent risk factors for later AV reintervention. After exclusion of patients with worse than mild AV regurgitation immediately after repair, both techniques functioned comparably (P = 0.089). CONCLUSIONS: AV reimplantation was associated with better valve function in longer-term postoperatively than remodelling. If optimal immediate repair outcome was achieved, both techniques provided comparable AV function.

A simple non-invasive method to collect soft tick saliva reveals differences in Ornithodoros moubata saliva composition between ticks infected and uninfected with Borrelia duttonii spirochetes.

Introduction: We developed a new simple method to assess the composition of proteinaceous components in the saliva of Ornithodoros moubata, the main vehicle for pathogen transmission and a likely source of bioactive molecules acting at the tick-vertebrate host interface. To collect naturally expectorated saliva from the ticks we employed an artificial membrane feeding technique using a simple, chemically defined diet containing phagostimulants and submitted native saliva samples collected in this way for liquid chromatography-mass spectrometry (LC-MS) analysis. These experiments were conducted with groups of uninfected ticks as well as with O. moubata infected with B. duttonii. The ticks exhibited a fair feeding response to the tested diet with engorgement rates reaching as high as 60-100% of ticks per feeding chamber. The LC-MS analysis identified a total of 17 and 15 proteins in saliva samples from the uninfected and infected O. moubata nymphs, respectively. Importantly, the analysis was sensitive enough to detect up to 9 different proteins in the samples of saliva containing diet upon which as few as 6 nymphal ticks fed during the experiments. Some of the proteins recognized in the analysis are well known for their immunomodulatory activity in a vertebrate host, whereas others are primarily thought of as structural or "housekeeping" proteins and their finding in the naturally expectorated tick saliva confirms that they can be secreted and might serve some functions at the tick-host interface. Most notably, some of the proteins that have long been suspected for their importance in the vector-pathogen interactions of Borrelia spirochetes were detected only in the samples from infected ticks, suggesting that their expression was altered by the persistent colonization of the tick's salivary glands by spirochetes. The simple method described herein is an important addition to the toolbox available to study the vector-host-pathogen interactions in the rapidly feeding soft ticks.

Unicuspid Aortic Valve in Patients Undergoing the Ross Procedure.

BACKGROUND: The prevalence and outcomes of the unicuspid aortic valve (UAV) in patients undergoing the Ross procedure have been strongly underreported in the current literature. We sought to evaluate this in comparison with bicuspid (BAV) and tricuspid valve (TAV) in our Ross cohort. METHODS: This was a retrospective observational study of patients undergoing the Ross procedure at 2 dedicated centers between 2009 and 2020. Primary end points were the risks of midterm autograft reoperation and the onset of at least moderate aortic regurgitation during follow-up. The secondary end point was to compare the perioperative outcomes between the groups. RESULTS: Included in the analysis were 286 patients, of those 39% with UAV, 52% with BAV, and 9% with TAV. UAV patients were operated on at the youngest age (P < .001) and more often for a combined hemodynamic aortic valve pathology (P = .02). They had the largest aortic root dimensions: annulus (P = .01), Valsalva sinuses (P = .11), sinotubular junction (P = .001), and ascending aorta (P < .0001). The risks of reoperation (P = .86) and the onset of aortic regurgitation (P = .75) were comparable among the groups over the follow-up of 4.1 years. There was no difference in perioperative outcomes. CONCLUSIONS: UAV is a separate unit characterized by a distinct hemodynamic pathology and generated aortopathy. It is not associated with a higher risk of reoperation or new onset of aortic regurgitation after the Ross procedure in the midterm postoperatively. At the current state, UAV remains acceptable for the Ross procedure.

Negligible risk of surface transmission of SARS-CoV-2 in public transportation.

BACKGROUND: Exposure to pathogens in public transport systems is a common means of spreading infection, mainly by inhaling aerosol or droplets from infected individuals. Such particles also contaminate surfaces, creating a potential surface-transmission pathway. METHODS: A fast acoustic biosensor with an antifouling nano-coating was introduced to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on exposed surfaces in the Prague Public Transport System. Samples were measured directly without pre-treatment. Results with the sensor gave excellent agreement with parallel quantitative reverse-transcription polymerase chain reaction (qRT-PCR) measurements on 482 surface samples taken from actively used trams, buses, metro trains and platforms between 7 and 9 April 2021, in the middle of the lineage Alpha SARS-CoV-2 epidemic wave when 1 in 240 people were COVID-19 positive in Prague. RESULTS: Only ten of the 482 surface swabs produced positive results and none of them contained virus particles capable of replication, indicating that positive samples contained inactive virus particles and/or fragments. Measurements of the rate of decay of SARS-CoV-2 on frequently touched surface materials showed that the virus did not remain viable longer than 1-4 h. The rate of inactivation was the fastest on rubber handrails in metro escalators and the slowest on hard-plastic seats, window glasses and stainless-steel grab rails. As a result of this study, Prague Public Transport Systems revised their cleaning protocols and the lengths of parking times during the pandemic. CONCLUSIONS: Our findings suggest that surface transmission played no or negligible role in spreading SARS-CoV-2 in Prague. The results also demonstrate the potential of the new biosensor to serve as a complementary screening tool in epidemic monitoring and prognosis.

Shotgun proteomic profiling of dormant, 'non-culturable' Mycobacterium tuberculosis.

Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by 'non-culturability'-a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, 'non-culturable' M. tuberculosis cells in an in vitro model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions-the raw data are available via ProteomeXchange with identifier PXD028849. This approach revealed the preservation of 1379 proteins in cells after 5 months of storage in dormancy; among them, 468 proteins were statistically different from those in the actively growing cells and bore a positive fold change (FC). Differential analysis revealed the proteins of the pH-dependent regulatory system PhoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages: especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current in vitro model of M. tuberculosis self-acidification reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins displaying antigenic properties makes it possible to distinguish immunoreactive proteins among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Ross procedure provides survival benefit over mechanical valve in adults: a propensity-matched nationwide analysis.

OBJECTIVES: The choice of optimal surgical treatment for young and middle-aged adults with aortic valve disease remains a challenge. Mechanical aortic valve replacement (mAVR) is generally preferred despite promising recent outcomes of the Ross procedure. Our goal was to compare the strategies at a nationwide level. METHODS: This study was a retrospective analysis of prospectively recorded data from the National Registry of Cardiac Surgery of the Czech Republic. Using propensity score matching, we compared the outcomes of patients undergoing the Ross procedure in 2 dedicated centres with all mAVRs performed in country between 2009 and 2020. RESULTS: Throughout the study period, 296 adults underwent the Ross procedure and 5120 had an mAVR. We found and compared 291 matched pairs. There were no in-hospital deaths, and the risk of perioperative complications was similar in both groups. Over the average follow-up period of 4.1 vs 6.1 years, the Ross group had a lower all-cause mortality (0.7 vs 6.5%; P = 0.015). This result remained significant even when accounting for cardiac- and valve-related deaths only (P = 0.048). Unlike the Ross group, the mAVR group had a significantly lower relative survival compared with the age- and sex-matched general population. There was no difference in the risk of reoperation (4.5 vs 5.5%; P = 0.66). CONCLUSIONS: The Ross procedure offers a significant midterm survival benefit over mAVR. The procedures have a comparable risk of perioperative complications. Patients after mAVR have reduced survival. Thus, the Ross procedure should be the preferred treatment option for young and middle-aged adults with aortic valve disease in dedicated centres.

Resonance assignment and secondary structure of DbpA protein from the European species, Borrelia afzelii.

Decorin binding proteins (Dbps) mediate attachment of spirochetes in host organisms during the early stages of Lyme disease infection. Previously, different binding mechanisms of Dbps to glycosaminoglycans have been elucidated for the pathogenic species Borrelia burgdorferi sensu stricto and B. afzelii. We are investigating various European Borrelia spirochetes and their interactions at the atomic level using NMR. We report preparative scale recombinant expression of uniformly stable isotope enriched B. afzelii DbpA in Escherichia coli, its chromatographic purification, and solution NMR assignments of its backbone and sidechain 1H, 13C, and 15N atoms. This data was used to predict secondary structure propensity, which we compared to the North American B. burgdorferi sensu stricto and European B. garinii DbpA for which solution NMR structures had been determined previously. Backbone dynamics of DbpA from B. afzelii were elucidated from spin relaxation and heteronuclear NOE experiments. NMR-based secondary structure analysis together with the backbone dynamics characterization provided a first look into structural differences of B. afzelii DbpA compared to the North American species and will serve as the basis for further investigation of how these changes affect interactions with host components.

Functionalized Terpolymer-Brush-Based Biointerface with Improved Antifouling Properties for Ultra-Sensitive Direct Detection of Virus in Crude Clinical Samples.

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.

Biotyping of IRE/CTVM19 tick cell line infected by tick-borne encephalitis virus.

Ticks, being vectors for a variety of pathogens such as tick-borne encephalitis virus (TBEV), have developed defense mechanisms and pathways against infections, allowing them to control the virus at a level that does not hinder their fitness and development. At the present moment, only a few studies focused on interactions between ticks and TBEV on a molecular level have been published. Here, a possible application of MALDI-TOF MS as a research tool for the investigation of tick-virus interactions was shown. Mass spectrometry (MS) profiles of TBEV-infected and non-infected IRE/CTVM19 tick cell line were compared using principal component analysis. MS spectra were clustered based on the cultivation time of cells, but not their infection status. Nevertheless, the analysis of loading plots revealed different factors (peaks) being involved in the clustering of infected and non-infected cells. Out of them, nine were assigned with proteins: five and four for non-infected and infected cells, respectively. Peak with m/z 8565 was found to be of interest because it was suppressed upon TBEV infection and assigned to proteasome subunit alpha type (B7QE67).